Method for determining susceptibility to escherichia coli urinary tract infections and method for diagnosing secretors and nonsecretors

ABSTRACT

A method for determining susceptibility to E. coli urinary tract infection comprising assaying a sample of epithelial cells for the presence or absence of at least one of sialosyl galactosyl-globoside, disialosyl galactosyl-globoside and an extended globo structure carrying the same terminal epitopes as sialosyl galactosyl-globoside or disialosyl galactosyl-globoside, or assaying a sample of vaginal secretions for the presence or absence of at least one of sialosyl galactosyl-globoside or disialosyl galactosyl-globoside, and detecting the presence or absence of the at least one of sialosyl galactosyl-globoside, disialosyl galactosyl-globoside and the extended globo structure, a medicament comprising a biologically effective amount of at least one E. coli bacterial receptor analogue and a pharmaceutically acceptable diluent, carrier or excipient as well as a method for preventing E. coli urinary tract infection comprising administering to a host a biologically effective amount of at least one E. coli bacterial receptor or bacterial receptor analogue are disclosed.

FIELD OF THE INVENTION

The present invention relates to a method for determining susceptibilityto Escherichia coli urinary tract infections and corresponding methodsfor diagnosing secretors and nonsecretors of histo-blood group antigensbased on assaying vaginal epithelial cells, vaginal secretions and/orbuccal epithelial cells. Also, the present invention relates to a methodfor preventing E. coli urinary tract infections as well as medicamentsused for prevention of E. coli urinary tract infections. In particular,the present inventors have discovered the importance of disialosylgalactosyl-globoside for the determination of secretor status as well aspreventing E. coli urinary tract inventions.

BACKGROUND OF THE INVENTION

Among the most common bacterial infections encountered in clinicalpractice are urogenital mucosal infections caused by E. coli, Chlamydiatrachomatis and N. gonorrheae which are diagnosed and treated at a costto society in the billions of dollars. These infections are frequentlyrecurrent and efforts at prevention have been largely fruitless, in partbecause of the apparent ineffectiveness of the mucosal immune system.Mucosal immunity is short-lived and in the long term, not protective.Despite much effort, attempts to develop vaccines against these mucosalurogenital pathogens have not succeeded and even natural immunity doesnot protect against recurrent episodes of infection with these agents.In addition, antigenic variability of the organisms has complicatedvaccine development.

In particular, acute uncomplicated urinary tract infections occur inmillions of young women each year. While most of these women experienceonly single or sporadic infections, approximately 20% suffer veryfrequent (≧3/year) recurrences (Mabeck, C.E., Postgrad. Med. J.,48:69-75, 1972). The apparent increased susceptibility to urinary tractinfection in these patients cannot be explained by underlying functionalor anatomic abnormalities of the urinary tract, but instead appears toarise from the interaction of infecting E. coli strains with thesepatients' epithelial cells.

Thus, women prone to frequent recurrences demonstrate prolongedcolonization of the vaginal mucosa with E. coli, the predominantcausative species in these infections, (Stamey, T.A. et al., J. Urol,113:214-217, 1975) and three-fold more E. coli adhere to vaginal, buccaland uroepithelial cells from women with recurrent urinary tractinfection than to cells from control patients (Schaeffer, A.J. et al.,N. Engl. J. Med., 304:1062-1066, 1981; Svanborg-Eden, C. et al., Infect.Immun., 26:837-840, 1979). Women with a history of recurrent urinarytract infections are also more likely to be nonsecretors of histo-bloodgroup antigens than are women without a history of infections (relativerisk=3 to 4 (Kinane, D.F. et al., Br. Med. J., 285:7-9, 1982; Sheinfeld,J. et al., N. Engl. J. Med., 320:773-777, 1989; Hooton, T.M. et al.,Twenty-ninth Interscience Conference on Antimicrobial Agents andChemotherapy, 8, 1989 (Program and Abstracts); Lomberg, H. et al.,Infect. Immun., 51:919-926, 1986)), and E. coli adhere in greaternumbers to uroepithelial cells from nonsecretors (Lomberg, H. et al.,Infect. Immun., 51:919-926, 1986).

E. coli adhesins also play an important role in the pathogenesis ofurinary tract infection (Stapleton, A. et al., J. Infect Dis.,163:773-779, 1989; Johnson, J.R., J. Infect. Dis., 156:225-229, 1987;Jacobson, S.H. et al., J. Infect. Dis., 152:426-427, 1985; Vaisenen, V.et al., Lancet, 2:1366-1369, 1981; Kallenius, G. et al., Lancet,2:1369-1372, 1981; Vaisanen-Rhen, V. et al., Infect. Immun., 48:149-155,1984; Westerlund, B. et al., J. Infect. Dis., 158:996-1002, 1988;Arthur, M. et al., Infect. Immun., 57:303-313, 1989; Sandberg, T., B.Kaijser, J. Clin. Microbiol, 26:1471-1476, 1988; O'Hanley, P. et al., N.Engl. J. Med., 313:414-420, 1985; Lidefelt, K.-J. et al., Acta Paediatr.Scand., 76:775-780, 1987; Lund, B. et al., Mol. Microbiol., 2:255-263,1988), particularly the genetically related P and F adhesins.

P adhesins are present in 50% to 65% of E. coli strains causing cystitis(Stapleton, A. et al., J. Infect Dis., 163:773-779, 1989; Arthur, M. etal., Infect. Immun., 57:303-313, 1989; Sandberg, T., B. Kaijser, J.Clin. Microbiol, 26:1471-1476, 1988; O'Hanley, P. et al., N. Engl. J.Med., 313:414-420, 1985; Lidefelt, K.-J. et al., Acta Paediatr. Scand.,76:775-780, 1987; Lund, B. et al., Mol. Microbiol., 2:255-263, 1988) and75% to 90% of isolates from pyelonephritis (Johnson, J.R., J. Infect.Dis., 156:225-229, 1987; Jacobson, S.Het et al., J. Infect. Dis.,152:426-427, 1985; Vaisenen, V. et al., Lancet, 2:1366-1369, 1981;Kallenius, G. et al., Lancet, 2:1369-1372, 1981; Vaisanen-Rhen, V. etal., Infect. Immun., 48:149-155, 1984; Westerlund, B. et al., J. Infect.Dis., 158:996-1002, 1988; Arthur, M. et al., Infect. Immun., 57:303-313,1989; Sandberg, T., B. Kaijser, J. Clin. Microbiol, 26:1471-1476, 1988).The minimal receptor for the P adhesin is the galactose α1-4 galactosemoiety, present in the globoseries glycolipids and the P₁ blood groupantigen (Kallenius, G. et al., FEMS Microbiol Lett., 7:297-302, 1980;Leffler, H. et al., FEMS Microbiol. Lett., 8:127-134, 1980;Svanborg-Eden, C. et al., Scand. J Infect Dis. Suppl., 24:144-147, 1980;Kallenius, G. et al., Infection, 8 (Suppl. 3):S288-S293, 1981;Kallenius, G- et al., Lancet, 2:604-606, 1981; Leffler, H. et al.,Infect Immun., 34:920-929, 1981; Kallenius, G. et al., Scand. J. Infect.Dis. Suppl., 33:52-60, 1982; Svenson, S. B. et al. , Infection,11:73/61- 79/67, 1983; Bock, K. et al., J. Biol Chem., 260:8545-8551,1985).

F adhesins are expressed in 30% to 65% of urinary tract infectionisolates (Stapleton, A. et al., J. Infect Dis., 163:773-779, 1989;Arthur, M. et al., Infect. Immun., 57:303-313, 1989). The minimalbinding moiety for the F adhesin is less well defined and probably morecomplex; proposed receptors include the Forssman and para-Forssmanantigens, globoside, galactosyl globoside, globo A and globo H, andstage-specific embryonic antigen-4 (SSEA-4) (Lund, B- et al., Mol.Microbiol., 2:255-263, 1988; Lindstedt, R. et al., Infect. Immun.,57:3389-3394, 1989; Karr, J.F. et al., Infect Immun., 58:4055-4062,1990; Stromberg, N. et al., EMBO J., 9:2001-2010, 1990). The globoseriesglycolipids have recently been shown to be modified by the histo-bloodgroup status, and ABH active globoseries GSLs are found in renalepithelium and in voided uroepithelial cells as well as on erythrocytes(Leffler, H. et al., FEMS Microbiol. Lett., 8:127-134, 1980; Bock, K. etal., J. Biol Chem., 260:8545-8551, 1985; Breimer, M.E. et al., FEBSLett, 179:165-172, 1985; Breimer, M.E. et al., J. Biochem.,98:1169-1180, 1985; Lindstedt, R. et al., Infect. Immun., 59:1086-1092,1991; Clausen, H. et al., Vox Sang., 56:1-20, 1989; Kannagi, R. et al.,FEBS Lett., 175:397-401, 1984; Clausen, H. et al., Biochem. Biophys.Res. Commun., 124:523-529, 1984).

Several lines of evidence have suggested that the increasedsusceptibility to recurrent urinary tract infection observed in someotherwise healthy women may be explained by genetic factors influencingthe density and/or specificity of bacterial receptors available tomediate colonization of their uroepithelial cells (Schaeffer, A.J. etal., N. Engl. J. Med., 304:1062-1066, 1981; Svanborg-Eden, C. et al.,Infect. Immun., 26:837-840, 1979; Kinane, D.F. et al., Br. Med. J.,285:7-9, 1982; Sheinfeld, J. et al., N. Engl. J. Med., 320:773-777,1989; Hooton, T.M. et al., Twenty-ninth Interscience Conference onAntimicrobial Agents and Chemotherapy, 8, 1989 (Program and Abstracts);Lomberg, H. et al., Infect. Immun., 51:919-926, 1986). Although it isknown that nonsecretors of histo-blood group antigens areoverrepresented among women with a history of recurrent urinary tractinfections (Kinane, D.F. et al., Br. Med. J., 285:7-9, 1982; Sheinfeld,J. et al., N. Engl. J. Med., 320:773-777, 1989; Hooton, T.M. et al.,Twenty-ninth Interscience Conference on Antimicrobial Agents andChemotherapy, 8, 1989 (Program and Abstracts)) and that uroepithelialcells from nonsecretors show enhanced E. coli adherence compared withcells from secretors (Lomberg, H. et al., Infect. Immun., 51:919-926,1986), the biochemical basis for these observations has not beenclarified.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to determine thebiochemical basis for the overrepresentation of nonsecretors among womenwith a history of recurrent E. coli urinary tract infections. Anotherobject of the present invention is to provide a method for determinationof susceptibility to E. coli urinary tract infections as well as methodsfor diagnosing secretors and nonsecretors. Correspondingly, an object ofthe present invention is to provide a method for prevention of E. coliurinary tract infections as well as a medicament to prevent theseurinary tract infections.

Accordingly, the present invention relates to a method for determiningsusceptibility to E. coli urinary tract infection comprising assaying asample of epithelial cells for the presence or absence of at least oneof Lewis^(a) (Le^(a)), sialosyl galactosyl-globoside (sialosylgal-globoside or SGG), disialosyl galactosyl-globoside (disialosylgal-globoside or DSGG) and an extended globo structure carrying the sameterminal epitopes as Le^(a), SGG or DSGG or assaying a sample of vaginalsecretions for the presence or absence of at least one of SGG or DSGG,and detecting the presence or absence of the at least one of Le^(a),SGG, DSGG and the extended globo structure.

The present invention further relates to methods for diagnosingnonsecretor and secretor status, that is, a method for diagnosingsecretors and nonsecretors of histo-blood group antigens comprisingassaying a sample of vaginal epithelial cells, vaginal secretions orbuccal epithelial cells for the presence or absence of at least one ofSGG and DSGG and detecting the presence or absence of the at least oneof SGG and DSGG and a method for diagnosing secretors of histo-bloodgroup antigens comprising assaying a sample of vaginal epithelial cellsor vaginal secretions for the presence or absence of at least one ofglobo H, globo ABO and lacto ABO, and detecting for the presence orabsence of the at least one of globo H, globo ABO and lacto ABO.

The present invention also relates to a medicament comprising abiologically effective amount of at least one E. coli bacterial receptoranalogue, and a pharmaceutically acceptable diluent, carrier orexcipient as well as a method for preventing E. coli urinary tractinfection comprising administering, to a host, a biologically effectiveamount of at least one E. coli bacterial receptor or bacterial receptoranalogue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show E. coli R45 binding to glycolipids extracted fromvaginal epithelial cells. FIG. 1A depicts a representative autoradiogramof bacterial binding to glycosphingolipids (GSL's) and FIG. 1B depictsan orcinol-stained HPTLC of the samples.

FIG. 2 depicts immunostaining of glycolipids extracted from pooledvaginal epithelial cells of nonsecretors and secretors.

FIG. 3 shows the results of a radiointmunoassay which used a monoclonalantibody to SGG to assay glycolipids from nonsecretors. The dotted lineindicates SGG standard from human pancreas. The solid line indicatesband co-migrating with SGG eluted from TLC of nonsecretors' extractedvaginal epithelial cells. The dashed line indicates eluted bandco-migrating with DSGG from nonsecretors and the following GSLstandards: gal-globoside and DSGG from human pancreas, GD1a from bovinebrain, and disialosyl I from human placenta.

FIGS. 4A and 4B depict immunostaining of glycolipids extracted fromvaginal epithelial cells of individual nonsecretors and secretors byautoradiographs from antibody overlay assays.

FIG. 5 is a bar graph depicting the degree of immunofluorescent stainingof vaginal epithelial cells collected from individual nonsecretors andsecretors and stained with a monoclonal antibody (MAb) to SGG.

DETAILED DESCRIPTION OF THE INVENTION

Several lines of evidence have suggested that the increasedsusceptibility to recurrent urinary tract infection observed in someotherwise healthy women may be explained by genetic factors influencingthe density and/or specificity of bacterial receptors available tomediate colonization of their uroepithelial cells (Schaeffer, A.J. etal., N. Engl. J. Med., 304:1062-1066, 1981; Svanborg-Eden, C. et al.,Infect. Immun., 26:837-840, 1979; Kinane, D.F. et al., Br. Med. J.,285:7-9, 1982; Sheinfeld, J. et al., N. Engl. J. Med., 320:773-777,1989; Hooton, T.M. et al., Twenty-ninth Interscience Conference onAntimicrobial Agents and Chemotherapy, 8, 1989 (Program and Abstracts);Lomberg, H. et al., Infect. Immun., 51:919-926, 1986). Although it isknown that nonsecretors of histo-blood group antigens areoverrepresented among women with a history of recurrent urinary tractinfections (Kinane, D.F. et al., Br. Med. J., 285:7-9, 1982; Sheinfeld,J. et al., N. Engl. J. Med., 320:773-777, 1989; Hooton, T.M. et al.,Twenty-ninth Interscience Conference on Antimicrobial Agents andChemotherapy, 8, 1989 (Program and Abstracts)) and that uroepithelialcells from nonsecretors show enhanced E. coli adherence compared withcells from secretors (Lomberg, H. et al., Infect. Immun., 51:919-926,1986), the biochemical basis for these observations has not beenclarified. The present invention is based upon the experiments describedbelow that show that nonsecretors express two unique GSL receptors forE. coli, SGG and DSGG, on their vaginal epithelial cells, which were notexpressed in secretors' vaginal epithelial cells. SGG was accessible tobinding by MAbs on the surface of the cells. Testing of the surfaceaccessibility of DSGG to MAb binding could not be conducted because ofthe lack of an appropriate MAb.

In the experiments, the following methods were used.

Preparation of Glycolipids

Vaginal epithelial cells were collected from healthy female collegestudents whose secretor statuses were determined using salivahemagglutination inhibition assays and whose red blood cell Le^(a)antigen phenotypes were determined using hemagglutination assays with anantibody to the Le^(a) antigen (Hooton, T.M. et al., Twenty-ninthInterscience Conference on Antimicrobial Agents and Chemotherapy, 8,1989 (Program and Abstracts)). Cells were collected by saline rinsingand gentle scraping with a spatula, were washed 4 times in phosphatebuffered saline (PBS), pH 7.3 and were stored in a freezing medium (85%M199 (Sigma), 10% fetal calf serum, 5% DMSO) at -70° C. until use(Hooton, T.M. et al. , Twenty-ninth Interscience Conference onAntimicrobial Agents and Chemotherapy, 8, 1989 (Program and Abstracts);Daifuku, R. et al., New Engl J. Med., 314: 1208-1213, 1986). Prior toglycolipid extraction, the cells were washed 4 times in PBS, quantitatedin a hemocytometer and equalized for extraction procedures. The cellsconstituted a homogeneous population possessing typical morphology asviewed on light microscopy. 6 to 8×10⁷ cells pooled from multiplepatients or 10⁷ cells/individual patient were used in each TLC assay.The total upper and lower phase glycolipid fractions were obtained asfollows: cells were extracted twice with 10 volumesisopropanol:hexane:water (IHW) (55:25:20 by volume) with sonication in awarm bath and centrifugation at 2,500 RPM for 10 minutes. The combinedsupernatants were dried under nitrogen and twice resuspended inchloroform:methanol (CM) (2:1 by volume) with one-sixth volume water,inverted 20 times, and centrifuged at 2,000 RPM for 10 minutes (Folch,J. et al., J. Biol Chem., 226:497-509, 1957). Total upper and lowerphases were then evaporated under nitrogen stream and resuspended in IHWfor chromatography.

For separation of total upper neutral glycolipids and gangliosides,upper phase glycolipids were first resuspended in 0.1% KCl in water,subjected to C18 Sep-Pak reverse phase column chromatography, washedwith water, eluted with methanol, dried, and passed over a DEAE SephadexA-25 column. Gangliosides were then eluted with 0.45M ammonium acetatein methanol, dried, and passed over a C18 column.

Glycolipids were preparatively separated by chromatography on glassHPTLC plates (Whatman) in chloroform:methanol:water (CMW) (50:40:10 byvolume) with 0.05% CaCl₂. The bands were visualized with primuline underUV light, marked with a pencil, scraped from the silica plates,extracted twice in IHW, and dried. Glycolipid standards were isolated byknown methods. To generate sialosyl gal-globoside, disialosylgal-globoside standard was partially desialylated with 1% acetic acid at100° C. for 5 to 10 minutes.

Bacterial Labelling

E. coli R45 was isolated from a woman with cystitis which had the papfamily genotype, expressing both the P and F adhesins (Stapleton, A. etal., J. Infect Dis., 163:773-779, 1989). Organisms were kept frozen in50% glycerol/50% Luria broth (Maniatis, T. et al., Molecular Cloning: ALaboratory Manual, 1982) at -70° C. until the night before use, whenthey were streaked onto Luria agar plates (Maniatis, T. et al.,Molecular Cloning: A Laboratory Manual, 1982) to promote fimbrialexpression and grown overnight at 37° C. On the day of use, bacteriawere scraped from the plate, resuspended in M9 medium (Maniatis, T. etal., Molecular Cloning: A Laboratory Manual, 1982) without amino acids,and shaken at 37° C. for 40 minutes to deplete cellular methioninestores. ³⁵ S-methionine (Trans³⁵ S-label, 1137 Ci/mmol, ICNBiochemicals, Irvine, Calif.) was then added at 200 μCi per 10¹⁰organisms. The cells were shaken for one hour, washed three times in PBSand resuspended in PBS (pH 7.3), and counted in a scintillation counterto give a final activity of approximately 0.01 to 0.02 cpm/organism.

Bacterial Overlay Assays

Glycolipids (10 to 40 μg/lane) were chromatographed on glass HPTLCplates (Whatman) in CMW (50:40:10 by volume) with 0.05% CaCl₂ with oneplate run in parallel for orcinol staining. Plates were dried, dippedfor 1 minute in diethyl ether containing 0.5% polyisobutylmethacrylate,dried, and preincubated in 1% bovine serum albumin (BSA)/PBS for onehour, then washed three times in PBS. Radiolabelled bacteria wereoverlaid (10⁸ cpm total per plate) and the plates were gently rocked forone hour, washed four times in PBS and subjected to autoradiography.

Antibody Overlay Assays and Radioimmunoassays of Glycolipids

Glycolipids separated on HPTLC were immunostained according to theprocedure of Magnani et al. (Magnani, J. L. et al., Anal Biochem.,109:399-402, 1980) as modified by Kannagi et al. (Kannagi, R. et al., J.Biol. Chem., 257:14865-14872, 1982).

Radioimmunoassays of glycosphingolipids (GSL's) microtiter wells wereperformed according to the method of Karlsson (Karlsson, K.-A. et al.,Methods Enzymol., 138:220-232, 1987). Specificities and sources of MAbsare given in Table 1.

Samples of patients having a susceptibility to E. coli urinary tractinfections, especially recurrent E. coli urinary tract infections,stained positive for Le^(a) and SGG.

                  TABLE 1                                                         ______________________________________                                        Specificities of Monoclonal Antibodies                                                     Monoclonal                                                       Antigen      Antibody    Reference                                            ______________________________________                                        Lewis.sup.a  CA3F4       American Type Culture                                                         Collection                                           Sialosyl gal-globoside                                                                     ID4         produced by                                                                   conventional methods                                 Disialosyl I NUH2        Nudelman, E. D. et al.,                                                       J. Biol. Chem.,                                                               264:18719-18725, 1989                                A type I, II, III                                                                          AH16        Abe, K. et al., J.                                                            Immunol.,                                                                     132:1951-1954, 1984                                  A type III, IV                                                                             HH5         Bremer, E. G. et al., J.                                                      Biol. Chem.,                                                                  259:14773-14777, 1984*                               Rabbit 1A    CRL1760     American Type                                                                 Culture Collection                                   ______________________________________                                         *monoclonal antibody (MAb) HH5 has the same antigenic specificity as MAb      MBr1, described in the reference.                                        

Immunofluorescence Staining of Vaginal Epithelial Cells

Vaginal epithelial cells were washed three times in PBS (pH 7.3),counted in a hemocytometer, and approximately 3×10⁴ cells wereresuspended in PBS. Cells were incubated on ice or at room temperaturewith the primary MAb or no antibody for one hour, washed 3 times in PBS,and incubated with the FITC-conjugated secondary antibody (diluted1:100) on ice for 30 minutes. Primary MAbs were undiluted ID4 or one oftwo control MAbs to unrelated antigens: NUH2 (undiluted) or CRL1760(ATCC; undiluted). After 3 additional washes in PBS, stained cells wereevaluated in a blinded fashion by examining each field sequentiallyusing fluorescent microscopy then light microscopy. Cells with faint orno staining were scored as unstained and all others were consideredpositive.

Samples of patients having a susceptibility to E. coli urinary tractinfections stained positive for SGG.

Using the above methods, the following experimentation was conducted.

Binding of E. coli R45 to Glycosphingolipid (GSL) Standards

The binding of metabolically radiolabelled E. coli strain R45 toglycolipid standards separated on HPTLC plates was determined using abroad panel of GSLs with varying carbohydrate moieties, as shown inTable 2. As predicted from its genotype and phenotype (P and F adhesinpositive), the bacteria bound to globoseries GSLs containing the minimalpap-binding moiety gal α1-4 gal as well as several of the suggestedreceptors for the F adhesin, including globoside, gal-globoside, theForssman antigen, and globo A and H.

                                      TABLE 2                                     __________________________________________________________________________    Binding of Escherichia coli R45 to Glycosphingolipids (GSL's)                 __________________________________________________________________________    Symbol Structure                           Binding                            __________________________________________________________________________    CMH    Glcβ1--1cer                    -                                  CDH    Galβ1-4Glcβ1--1cer        -                                  CTM    Galα1-4Galβ1-4Glcβ1--1cer                                                                         +                                  Globoside                                                                            GalNAcβ1-3Galα1-4Galβ1-4Glcβ1--1cer                                                          +                                  Gal-globoside                                                                        Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1--1ce           r                                   +                                  Forssman                                                                             GalNAcα1-3Galβ1-3GalNAcβ1-3Galα1-4Galβ1-           4Glcβ1--1cer                   +                                  Globo H                                                                              (Fucα1-2)Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4           Glcβ1--1cer                    +                                  Globo A                                                                              GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcβ1-3Gal.alpha           .1-4Galβ1-4Glcβ1--1cer    +                                  SGG    NeuAcα2-3Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4           Glcβ1--1cer                    +                                  DSGG   NeuAcα2-3(NeuAcα2-6)Glaβ1-3GalNAcβ1-3Gal.alph           a.1-4Galβ1-4Glcβ1--1cer   +                                  ASGM1  Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1--1cer                                                           -                                  ASGM2  GalNAcβ1-4Galβ1-4Glcβ1--1cer                                                                       -                                  nLc6   Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4G           lcβ1--1cer                     -                                  B1     Galα1-3(Fucα 1-2)Galβ1-3GlcNAcβ1-3Galβ1-           4Glcβ1--1cer                   -                                  __________________________________________________________________________    Abbreviations:                                                                CMH -- ceramide trihexoside;                                                  CDH -- ceramide dihexoside;                                                   CTH -- ceramide trihexoside (globotriaosylceramide);                          globoside -- globotetrasylceramide;                                           gal-globoside -- galactosyl globoside;                                        SGG -- sialosyl gal-globoside;                                                DSGG -- disialosyl gal-globoside;                                             ASGM1 -- asialo GM1;                                                          ASMG2 -- asialo GM2;                                                          nLc6 -- lacto-N-norhexaosylceramide;                                          B1 -- type 1 chain B antigen;                                                 Glc -- glucose;                                                               cer -- ceramide;                                                              Gal -- galactose;                                                             GalNAc -- N-acetylgalactosamine;                                              Fuc -- fucose;                                                                NeuAc -- neuraminic acid;                                                     GlcNAc -- N-acetylglucosamine.                                                __________________________________________________________________________

Binding of E. coli R45 to Glycolipids Extracted from Vaginal EpithelialCells

Total upper and lower phase GSLs were extracted from pooled vaginalepithelial cells collected from 3 groups of healthy college students: 10nonsecretors (8 with a history of recurrent urinary tract infection and2 without); 12 secretors with a history of recurrent urinary tractinfection; and 18 secretors without recurrent infections. Thedistribution of ABO histo-blood group phenotypes among the patients inthe groups was comparable. E. coli strain R45 (isolated from the urineof one of the patients with a urinary tract infection) was metabolically³⁵ S-labelled as described above and reacted with the total upper andlower phase GSLs from each of the 3 groups of patients in bacterialHPTLC overlay assays as described above.

The organism bound to a unique glycolipid band in the total upper phaseGSLs from nonsecretors which was not seen in GSLs from either group ofsecretors (data not shown). This experiment was repeated using pooledvaginal cells from 10 nonsecretors and 20 secretors (10 with and 10without a history of recurrent urinary tract infection) with identicalresults. No differences in binding were detected in either experimentwhen secretors with and without a history of recurrent urinary tractinfection were compared.

Subsequent experiments were conducted to identify the unique E.coli-binding GSL band detected in nonsecretors' vaginal epithelialcells. Total upper neutral GSLs and total upper gangliosides wereextracted from vaginal epithelial cells pooled in equal quantities from5 nonsecretors and 9 secretors and reacted with radiolabelled E. coliR45. The results are shown in FIGS. 1A and 1B.

FIG. 1A shows a representative autoradiogram of bacterial binding toGSLs extracted from equal quantities of pooled vaginal epithelial cellsfrom 5 nonsecretors and 9 secretors and to GSL standards. Anorcinol-stained HPTLC of the samples is shown in FIG. 1B.

Lane 1 contains ceramide trihexoasyl and globoside standards;

Lane 2 contains total lower phase GSLs from the nonsecretors;

Lane 3 contains total lower phase GSLs from the secretors;

Lane 4 contains the Forssman antigen standard;

Lanes 5 and 6 contain total upper neutral GSLs from nonsecretors andsecretors, respectively;

Lanes 7 and 10 contain gal-globoside, sialosyl gal-globoside, anddisialosyl gal-globoside standards;

Lanes 8 and 9 contain total upper phase gangliosides from nonsecretorsand secretors, respectively.

In total ganglioside fractions from nonsecretors, but not fromsecretors, the organism bound to bands co-migrating with SGG and DSGGstandards and also reacted with the standards themselves (FIG. 1A). Thetotal amount of glycolipid placed in each lane was normalized based uponequivalent numbers of cells. As can be seen from the orcinol TLC, thechemical amounts of total upper phase gangliosides from nonsecretors andsecretors are visually comparable (FIG. 1B, Lanes 8 and 9).

Immunostaining and Radioimmunoassays of Glycolipids Extracted fromPooled Vaginal Epithelial Cells

To confirm the identity of SGG in the total upper ganglioside fractionfrom nonsecretors' vaginal epithelial cells, bands co-migrating with SGGand DSGG were eluted from HPTLC plates, chromatographed along with totalgangliosides from nonsecretors and secretors, and reacted with MAb ID4directed against SGG.

The results are shown in FIG. 2 in which Lanes 1 and 2 contain totalgangliosides from nonsecretors and secretors, respectively. Lane 3contains the eluted band from nonsecretors which co-migrated with SGGand Lane 4 contains the eluted band from nonsecretors which co-migratedwith DSGG. Lanes 5 and 7 contains SGG standards from human pancreas andLane 6 contains gal-globoside standard from human pancreas (note traceof SGG).

As shown in FIG. 2, the MAb stained a band co-migrating with SGG in Lane1, containing the total upper ganglioside fraction from nonsecretorsand, in Lane 3, containing the putative SGG eluted from thenonsecretors. No staining was seen in the total ganglioside fractionfrom the secretors (Lane 2) nor in the putative DSGG material from thenonsecretors (Lane 4).

The SGG and DSGG bands from the nonsecretors' vaginal epithelial cellextracts were then reacted with ID4 in a radioimmunoassay, shown in FIG.3.

In FIG. 3, the dotted line indicates sialosyl gal-globoside (SGG)standard from human pancreas; the solid line indicates the bandco-migrating with SGG eluted from TLC of nonsecretors' extracted vaginalepithelial cells; the dashed line indicates eluted band co-migratingwith DSGG from nonsecretors and the following GSL standards:gal-globoside and DSGG from human pancreas, GD1a from bovine brain, anddisialosyl I from human placenta.

FIG. 3 shows that the antibody reacted with the SGG band fromnonsecretors and with the SGG standard but did not bind to thenonsecretor DSGG band nor to the DSGG, gal-globoside, GD1a, ordisialosyl I standards.

To determine if the nonsecretor phenotypes of the patients as determinedby blood and saliva testing was also expressed in vaginal epithelialcells, total upper phase GSLs from nonsecretors were chromatographed andreacted with MAbs "anti Le^(a) " and AH16 (against A type I, II andIII). In the nonsecretor GSLs, a band co-migrating with Le^(a) standardwas brightly stained but no staining was observed with MAb AH16 (datanot shown). In addition, MAbs against CTH and globoside/gal-globosidestained the cell extracts, confirming that globoseries GSLs areexpressed in vaginal epithelial cells (data not shown).

Immunostaining of Glycolipids Extracted from Vaginal Epithelial Cellsfrom Individual Patients

To determine whether SGG is found in all nonsecretors (and is absent inall secretors) or present only in some nonsecretors, the total upper andlower phase GSLs were extracted from vaginal epithelial cells collectedfrom 5 separate nonsecretor individuals as well as from 5 separatesecretor individuals. Equal quantities of cells (10⁷ cells/patient) wereextracted and chromatographed as described and reacted with antibodiesID4 (against SGG) and HH5 (against A type III, IV). The secretor, ABOand Le^(a) red blood cell phenotypes of the patients (determined byhemagglutination assays) are given in Table 3.

                  TABLE 3                                                         ______________________________________                                        Secretor, Lewis.sup.a and ABO Phenotypes and                                  Antibody Binding                                                                                                 Antibody                                   Secretor       ABO       Lewis.sup.a                                                                             Binding                                    Patient No.                                                                           Phenotype  Phenotype Phenotype                                                                             HH5  ID4                                 ______________________________________                                        1       Nonsecrotor                                                                              A         +       -    +                                           (NS)                                                                  2       NS         O         +       -    +                                   3       NS         O         +       -    +                                   4       NS         A         +       -    +                                   5       NS         B         +       -    +                                   6       Secretor (S)                                                                             O         -       -    -                                   7       S          O         -       -    -                                   8       S          AB        -       +    -                                   9       S          AB        -       +    -                                   10      S          A         -       +    -                                   ______________________________________                                    

The results are shown in FIG. 4, wherein Lanes 1-10, contain samples insequence from patients 1 to 10. Lane 11 in FIG. 4A contains SGG standardfrom human pancreas and in FIG. 4B contains upper neutral GSLs from typeA human red blood cells.

As shown in FIG. 4A, SGG was detected in total upper phase GSLs fromeach of the 5 nonsecretors (Lanes 1-5), but not from any of thesecretors Lanes 6-10).

In FIG. 4B, MAb HH5 detected A-reactive substances in GSLs from thethree secretors with A or AB phenotypes (Lanes 8, 9 and 10) but not fromthe nonsecretors (Lanes 1-5).

Immunofluorescence Assays with Native Vaginal Epithelial Cells

To demonstrate that SGG is present and accessible on the surface ofnative vaginal epithelial cells from nonsecretors, but not secretors,immunostaining of cells from 2 individual secretors and 2 nonsecretorsin three trials using MAb ID4 was compared. The results for the totalnumber of cells stained in the three assays combined are shown in FIG.5. Cells with faint or no staining were considered negative while anyother degree of staining was considered positive.

As shown in FIG. 5, the majority of nonsecretors' vaginal epithelialcells were stained, while little or no staining of secretors' cells wasobserved. Staining of cells from both patient groups with control MAbsNuH2 and CRL1760 (against unrelated antigens) was comparable andminimal.

The above experiments demonstrate binding moieties unique tononsecretors with structures consistent with Se gene-mediated control ofantigen expression in globoseries GSLs from vaginal epithelial cells.SGG and DSGG were detected in extracts of aliquoted vaginal epithelialcells pooled from a total of 10 nonsecretors and also in cells collectedand separately extracted from 5 additional individuals with the se/sephenotype, suggesting that the expression this antigen is uniform fornonsecretors. Considering all bacterial binding experiments using bothpooled and individuals' vaginal epithelial cells, a total of 35secretors' and 15 nonsecretors' epithelial cells were studied. The datasupports the hypothesis that the Se locus controls the expression of ABOblood group antigen variants of globoseries GSLs in vaginal epithelialcells, an anatomic site which plays an important role in the sequence ofcolonization events preceding the development of urinary tract infectionin women.

As described above, several globoseries GSLs are known receptors foruropathogenic E. coli and are present in other urinary tract sites(Lund, B. et al., Mol. Microbiol., 2:255-263, 1988; Kallenius, G. etal., FEMS Microbiol Lett., 7:297-302, 1980; Leffler, H. et al., FEMSMicrobiol. Lett., 8:127-134, 1980; Svanborg-Eden, C. et al., Scand. JInfect Dis. Suppl., 24:144-147, 1980; Kallenius, G. et al., Infection, 8(Suppl. 3):S288-S293, 1981; Kallenius, G. et al., Lancet, 2:604-606,1981; Leffler, H. et al., Infect Immun., 34:920-929, 1981; Kallenius, G.et al., Scand. J. Infect. Dis. Suppl., 33:52-60, 1982; Svenson, S.B. etal., Infection, 11:73/61-79/67, 1983; Bock, K. et al., J. Biol Chem.,260:8545-8551, 1985; Lindstedt, R. et al., Infect. Immun., 57:3389-3394,1989; Karr, J.F. et al., Infect Immun., 58:4055-4062, 1990; Stromberg,N. et al., EMBO J., 9:2001-2010, 1990; Breimer, M.E. et al., J.Biochem., 98:1169-1180, 1985; Lindstedt, R. et al., Infect. Immun.,59:1086-1092, 1991), but the influences of the Se gene on globoseriesbiosynthesis in vaginal epithelium has not been previously been known tohave been demonstrated. One study suggested that globoseries GSLsynthesis in voided uroepithelial cells from adults depended on ABO andsecretor status (Leffler, H. et al., In Host Parasite Interactions inUrinary Tract Infections, 93-99, 1989). The presence of SGG and DSGG inuroepithelial cells from nonsecretors may account for the increasedbinding of E. coli to their cells and for their increased susceptibilityto recurrent urinary tract infection.

In addition to supporting the role of a genetic host factor in thepathogenesis of recurrent urinary tract infection in women, the data ofthe present invention clarifies the mechanism by which this occurs.

Others have speculated that E. coli receptors on uroepithelial cellsfrom nonsecretors are more accessible because of the lack of fucosyltransferase-mediated synthesis of A, B and H antigens, whereas insecretors, the presence of histo-blood group antigens on epithelialcells might shield the receptors and prevent bacterial binding(Sheinfeld, J. et al., N. Engl. J. Med., 320:773-777, 1989; Lomberg, H.et al., Infect. Immun., 51:919-926, 1986; Schoolnik, G.K., New Engl. J.Med., 320:804-805, 1989). However, reports correlating secretor statuswith the expression of histo-blood group antigens on Type I-IVcarbohydrate core structures in the urinary tract are conflicting(Orntorft, T.F. et al., Lab. Invest., 58:576-583, 1988). The correlationof secretor state with expression of ABH antigens has only been clearlydemonstrated for antigens carried on Type I chains (Leffler, H. et al.,In Host Parasite Interactions in Urinary Tract Infections, 93-99, 1989;Orntorft, T.F. et al., Lab. Invest., 58:576-583, 1988), buturopathogenic E. coli have not been reported to bind to Type I core GSLs(Leffler, H. et al., In Host Parasite Interactions in Urinary TractInfections, 93-99, 1989).

This was also found to be true by the present inventors. In GSLsextracted from vaginal epithelial cells from nonsecretors and secretors,respectively, the present inventors identified major bands correspondingin TLC migration to Lewis^(a) and Lewis^(b) antigens. In extracts fromnonsecretors, the Le^(a) band reacted with a MAb directed to Le^(a).However, E. coli did not bind to either of the Lewis^(a) or Lewis^(b)bands. In contrast, the present inventors have shown that on vaginalepithelial cells, there is a correlation between secretor status and ABHantigen expression of well-described receptors for uropathogenic E.coli, the Type IV chain (globoseries) GSLs. Rather than failing toshield bacterial receptors, nonsecretors synthesize unique sialylated,E. coli binding derivatives of the globoseries GSLs.

Genetic variability in glycosylation encoded by blood group genes isgenerally thought to be involved in "masking" or shielding glycanreceptors for microbial pathogens. Globoseries GSLs characterized by aterminal or internal galactose alpha 1-4 galactose moiety are preferredreceptors for E. coli adhesins. Previous studies have clearly shown thatthe binding of E. coli to globoseries GSLs changes when the disaccharidereceptor site is further modified by elongation of the saccharide chain(Bock, K. et al., J. Biol Chem., 260:8545-8551, 1985). The geneticvariability inherent to blood group antigens implies that a proportionof the population lacks certain glycan structures. In such persons,competition for terminal glycosylation of the precursor gal-globoside isshared by both a fucosyltransferase and a sialyltransferase. Apparently,the affinity of the fucosyltransferase for the terminal galactose isgreater and hence the Globo H structure is synthesized to the exclusionof any terminally sialylated structures. In contrast, nonsecretors lacksuch a fucosyltransferase and synthesize SGG and DSGG in epithelialcells through sialylation of the precursor gal-globoside, with nocompetition for terminal fucosylation by the absent Se gene-encodedfucosyltransferase. As evidenced by the results of the experimentationleading to the present invention, this sialylation does not interferewith the receptor activity of the globo core.

In summary, the present inventors have shown that nonsecretors ofhisto-blood group antigens synthesize unique GSLs, SGG and DSGG, ontheir vaginal epithelial cells which are not found in cells fromsecretors. As these moieties serve as receptors for uropathogenic E.coli, this finding provides a biochemical explanation for the increasedadherence of bacteria to these women's uroepithelial cells and for theirpropensity to develop recurrent urinary tract infection. The presentinventors have also shown that other globoseries GSLs known to bereceptors for uropathogenic E. coli are present in GSL extracts fromvaginal epithelial cells and bind bacteria in HPTLC overlay assays. Innonsecretors, SGG and DSGG may be more abundant or accessible on vaginalepithelial cells than other E. coli receptors, or the affinity ofbacterial binding for these moieties may be higher than the affinity forother globoseries GSLS. Sialylation may alter the conformation of theadhesin binding site in favor of increased bacterial binding to SGG andDSGG as compared with binding to other available receptors.

Method for Determining Susceptibility to E. coli Urinary TractInfections

Accordingly, the present invention provides a method for determiningsusceptibility to E. coli urinary tract infection comprising assaying asample of epithelial cells for the presence or absence of at least oneof Le^(a), SGG, DSGG and an extended globo structure carrying the sameterminal epitopes as Le^(a), SGG, or DSGG or assaying a sample ofvaginal secretions for the presence or absence of at least one of SGG orDSGG, and detecting the presence or absence of the at least one ofLe^(a), SGG, DSGG and the extended globo structure.

In the method for determining susceptibility to E. coli urinary tractinfections, the assay is conducted by first collecting epithelial cellsor vaginal secretions, with the secretions constituting the firstsupernatant sample from washing the cells, from a patient and preparingglycolipids as described above.

The epithelial cells which can be collected include uroepithelial cells,vaginal epithelial cells and buccal epithelial cells.

The glycolipid samples can be assayed for Le^(a) and/or SGG by any knownimmunoassay method, preferably by an immunoblot assay, more preferablyby an antibody overlay assay, a bacterial overlay assay and/or aradioimmunoassay.

When an antibody or bacterial overlay assay is used, the bacteria can belabelled as described above and the antibody can be labelled accordingto known methods. The bacterial overlay assay and/or antibody overlayassay can be conducted as described above.

When a radioimmunoassay is used, the radioimmunoassay can be conductedas described above.

Any appropriate monoclonal antibodies can be used. These monoclonalantibodies are either publicly available or can be produced byconventional methods. Preferably a panel of monoclonal antibodies can beused in the assay as set forth in Table 1 above, that is, CA3F4, ID4,NUH2, AH16, HH5, and CRL1760. CA3F4 and CRL1760 are available from theAmerican Type Culture Collection. The other preferred monoclonalantibodies can be produced by conventional methods.

Detecting the presence of Le^(a), SGG, DSGG, and an extended globostructure carrying the same terminal epitopes as Le^(a), SGG, or DSGGafter assaying can be conducted in accordance with appropriate methodsknown in the art.

Samples of patients having a susceptibility to E. coli urinary tractinfections, especially recurrent E. coli urinary tract infections, willstain positive for Le^(a), SGG, DSGG and the extended globo structurecarrying the same terminal epitopes as Le^(a), SGG, or DSGG.

Methods for Diagnosing Secretors and Nonsecretors of Histo-Blood GroupAntigens

The present invention also provides a method for diagnosing secretorsand nonsecretors of histo-blood group antigens comprising assaying asample of vaginal epithelial cells, vaginal secretions or buccalepithelial cells for the presence or absence of at least one of SGG andDSGG, and detecting the presence or absence of the at least one of SGGand DSGG.

In the method for diagnosing nonsecretors and secretors of histo-bloodgroup antigens based on the presence of SGG and/or DSGG, the assay isconducted by first collecting vaginal epithelial cells, vaginalsecretions or buccal epithelial cells from a patient.

The cells or secretions can be treated in accordance with any knownmethod in preparation for assaying depending on the assay method to beused. Preferably, the cells or secretions are treated in accordance withthe method outlined above in preparation for immunofluorescencestaining, with the secretions constituting the first supernatant samplefrom washing the cells.

The samples can be assayed for SGG and/or DSGG by any known immunoassaymethod. Preferably, immunofluorescence or a colorimetric ELISA method isused.

Any appropriate monoclonal antibody known in the art can be used. Thesemonoclonal antibodies can be produced by conventional methods.Preferably, MAb ID4, which can be prepared by conventional methods, isused. Also, an antibody against stage-specific embryonic antigen-4(SSEA-4), which is presumed to have the same structure as SGG, isexpected to be used for detecting SGG and is known as MC813-70. MC813-70can be produced by conventional methods. Stage-specific embryonicantigens (SSEA-3 and SSEA-4) are epitopes of a unique globo-seriesganglioside isolated from human teratocarcinoma cells. Embo J., Vol. 2,pages 2355-2361, 1983. Appropriate Mabs for DSGG can also be made byconventional methods.

Detecting the presence or absence of SGG and/or DSGG after assaying canbe conducted in accordance with appropriate methods known in the art.When the detection is based on immunostaining of the cells, cells ofnonsecretors will be stained, whereas little or no staining will occurin cells of secretors.

The present inventor further provides a method for diagnosing secretorsof histo-blood group antigens comprises assaying a sample of vaginalepithelial cells or vaginal secretions for the presence or absence of atleast one of globo H, globo ABO and lacto ABO, and detecting for thepresence or absence of the at least one of globo H, globo ABO and lactoABO.

In the method for diagnosing secretors of histo-blood group antigensbased on the presence of globo H, globo ABO and lacto ABO, the assay isconducted by first collecting vaginal cells and/or secretions from apatient.

The cells and/or secretions are treated in accordance with any knownmethods in the art in preparation for assaying depending on the assaymethod to be used. Preferably, the cells and/or secretions are treatedin accordance with the method outlined above in preparation forimmunofluorescence staining, with the secretions constituting the firstsupernatant sample from washing the cells.

The samples can be assayed for globo H, globo ABO and/or lacto ABO byany known immunoassay method. Preferably, immunofluorescence or acolorimetric ELISA assay is used.

Any appropriate monoclonal antibody known in the art can be used and canbe produced by conventional methods.

Detecting the presence or absence of globo H, globo ABO and/or lacto ABOafter assaying can be conducted in accordance with appropriate methodsknown in the art. When the detection is based on immunostaining of thecells, cells of secretors will be stained, whereas little or no stainingwill occur in cells of nonsecretors.

Medicament for Preventing E. coli Urinary Tract Infections

The present invention further provides a medicament comprising abiologically effective amount of at least one bacterial receptoranalogue, and a pharmaceutically acceptable diluent, carrier orexcipient.

Preferably, the at least one bacterial receptor analogue is a stabilizedsynthetic carbohydrate. More preferred bacterial receptor analoguesinclude carbohydrates having thioglycosides, N-trifluoroacetyl- orN-carbamyl-sialic acid, and replacement of O with S in hexose ringstructures.

The at least one bacterial receptor analogue can be an analogue ofbacterial receptors including galactosyl-globoside, SGG, DSGG or globoH.

The bacterial receptors used in the present invention can be prepared bymethods which are known in the art.

The bacterial receptor analogues can be prepared by methods which areknown in the art.

The biologically effective amount of the medicament according to thepresent invention can be determined using art-recognized methods, suchas by establishing dose response curves in suitable animal models andextrapolating to humans; extrapolating from in vitro data; or bydetermining effectiveness in clinical trials.

Suitable doses of the receptor analogue according to the presentinvention depend upon the particular medical application, the severityof the condition, the half-life in circulation, etc., and can easily bedetermined by the skilled artisan.

The number of doses, daily doses and course of treatment may vary fromindividual to individual.

Depending on the particular medical application, the medicament can beadministered in a variety of ways such as orally and topically. Also,the medicament can be administered in a spermicidal preparation.

The selection of suitable pharmaceutically acceptable carriers, diluentsor excipients which can be used in the medicament for administrationdepends on the particular medical use and can be determined readily bythe skilled artisan.

The medicament can take a variety of forms, such as tablets, capsules,bulk or unit dose powders or granules; may be contained with liposomes;or may be formulated into solutions, emulsions, suspensions, ointments,pastes, creams, jells, foams or jellies and the like.

Additionally, a variety of art-recognized carriers, excipients,diluents, fillers, etc., are likely to be included in the dosage forms.Such subsidiary ingredients include disintegrants, binders, lubricants,surfactants, emulsifiers, buffers, moisturizers, solubilizers andpreservatives. The artisan can configure the appropriate formulationcomprising the compound seeking guidance from numerous authorities andreferences such as Goodman et al., Goodman & Gilman's, ThePharmaceutical Basis of Therapeutics, 6th Ed., 1980.

Method for Preventing E. coli Urinary Tract Infections

The present invention further provides a method for preventing E. coliurinary tract infection comprising administering to a host abiologically effective amount of at least one E. coli bacterial receptoror bacterial receptor analogue.

The at least one bacterial receptor can include galactosyl-globoside,SGG, DSGG and globo H and can be prepared in accordance with methodsknown in the art.

The at least one bacterial receptor analogue is as described for themedicament.

The bacterially effective amount can be determined as described abovefor the medicament.

The method is especially useful for preventing recurrent E. coli urinarytract infections.

EXAMPLES

The invention will now be described by reference to specific examples,which are merely exemplary and are not considered to limit theinvention.

Example 1

Assay for Determining Susceptibility to E. coli Urinary Tract Infection

Vaginal epithelial cells were collected from a patient population bysaline rinsing and gentle scraping. The cells were washed 4 times inphosphate buffered saline (PBS), pH 7.3 and were stored in a freezingmedium (85% M199 (Sigma), 10% fetal calf serum, 5% DMSO) at -70° C.until use. Prior to glycolipid extraction, the cells were washed 4 timesin PBS, quantitated in a hemocytometer and equalized for extractionprocedures. The total upper and lower phase glycolipid fractions wereobtained as follows: cells were extracted twice with 10 volumesisopropanol:hexane:water (IHW) (55:25:20 by volume) with sonication in awarm bath and centrifugation at 2,500 RPM for 10 minutes. The combinedsupernatants were dried under nitrogen and twice resuspended inchloroform: methanol (CM) (2:1 by volume) with one-sixth volume water,inverted 20 times, and centrifuged at 2,000 RPM for 10 minutes. Totalupper and lower phases were then evaporated under nitrogen stream andresuspended in IHW for chromatography.

For separation of total upper neutral glycolipids and gangliosides,upper phase glycolipids were first resuspended in 0.1% KCI in water,subjected to C18 Sep-Pak reverse phase column chromatography, washedwith water, eluted with methanol, dried, and passed over a DEAE SephadexA-25 column. Gangliosides were then eluted with 0.45M ammonium acetatein methanol, dried, and passed over a C18 column.

Glycolipids were preparatively separated by chromatography on glassHPTLC plates (Whatman) in chloroform:methanol:water (CMW) 50:40:10 with0.05% CaCl₂. The bands were visualized with primuline under UV light,marked with a pencil, scraped from the silica plates, extracted twice inIHW, and dried.

The glycolipid samples were then assayed for the presence of Le^(a) andSGG by antibody overlay assays and radioimmunoassay methods describedabove using a panel of MAbs set forth in Table 1.

Immunostaining was evaluated. Samples of patients having asusceptibility for E. coli urinary tract infections stained positive forLe^(a) and SGG.

Example 2

Assay for Determining Nonsecretors and Secretors of Histo-Blood GrougAntigens

Vaginal epithelial cells were collected from patients having secretorstatus and nonsecretor status with saline and gentle scraping with aspatula to obtain cell samples. (Secretor status and nonsecretor statuswere determined beforehand by using a hemagglutination inhibition assayperformed on saliva.) These cells were washed three times in PBS (pH7.3), counted in a hemocytometer, and approximately 3×10⁴ cells wereresuspended in PBS.

The samples were then assayed by immunofluorescence using MAb ID4 todetect the presence of SGG by incubating the cells on ice or at roomtemperature with the primary MAb ID4 for one hour, washed 3 times inPBS, and incubated with the FITC-conjugated secondary antibody (diluted1:100) on ice for 30 minutes. After 3 additional washes in PBS, stainedcells were evaluated in a blinded fashion by examining each fieldsequentially using fluorescent microscopy then light microscopy.

Immunostaining was then evaluated. Cells with faint or no staining werescored as unstained and all others were considered positive.

Samples of patients having nonsecretor status stained positive for SGG.

Example 3

Assay for Determining Secretors of Histo-Blood Group Antigens

Vaginal epithelial cells were collected from patients having secretorstatus and nonsecretor status with saline and gentle scraping with aspatula to obtain cell samples. These cells were washed three times inPBS (pH 7.3), counted in a hemocytometer, and approximately 3×10⁴ cellswere resuspended in PBS.

The samples were then assayed by immunofluorescence using available MAbsto detect the presence of globo H, globo ABO and lacto ABO by incubatingthe cells on ice or at room temperature with the primary MAb for onehour, washing 3 times in PBS, and incubating with an FITC-conjugatedsecondary antibody (diluted 1:100) on ice for 30 minutes. After 3additional washes in PBS, stained cells were evaluated in a blindedfashion by examining each field sequentially using fluorescentmicroscopy then light microscopy.

Immunostaining was then evaluated. Cells with faint or no staining werescored as unstained and all others were considered positive.

Samples of patients having secretor status stained positive for globo H,globo ABO and/or lacto ABO.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for determining susceptibility to E.coli urinary tract infection (IUTI) comprising:(a) assaying a sample ofepithelial cells for the presence or absence of at least one of sialosylgalactosyl-globoside (SGG), disialosyl galactosyl-globoside (DSGG) andan extended globo structure carrying the same terminal epitopes assialosyl galactosyl-globoside or disialosyl galactosyl-globoside, orassaying a sample of vaginal secretions for the presence or absence ofat least one of sialosyl galactosyl-globoside or disialosylgalactosyl-globoside, and (b) detecting the presence or absence of theat least one of sialosyl galactosyl-globoside, disialosylgalactosyl-globoside and the extended globostructure; wherein presenceof SGG, DSGG or extended globo structure carrying SGG and DSGG epitopescorrelates with an increased susceptibility to UTI.
 2. The method ofclaim 1, wherein the epithelial cells are selected from the groupconsisting of uroepithelial cells, vaginal epithelial cells and buccalepithelial cells.
 3. The method of claim 1, wherein the assaying isperformed by an assay selected from the group consisting of antibodyoverlay assays, bacterial overlay assay and radioimmunoassays.
 4. Themethod of claim 1, wherein the E. coli urinary tract infection isrecurrent.
 5. A method for diagnosing secretors and nonsecretors ofhisto-blood group antigens comprising:(a) assaying a sample of vaginalepithelial cells, vaginal secretions or buccal epithelial cells for thepresence or absence of at least one of sialosyl gal-globoside (SGG) anddisialosyl gal-globoside (DSGG), and (b) detecting the presence orabsence of the at least one of sialosyl gal-globoside and disialosylgal-globoside; wherein SGG and DSGG is found in samples fromnonsecretors.
 6. The method of claim 5, wherein the assaying isperformed by immunofluorescence or a colorimetric ELISA assay.